Synthesis and anticancer activity of new dihydropyrimidinone derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.07.004

A series of dihydropyrimidinone derivatives bearing various N-heterocyclic moieties was designed and synthesized. Twelve new compounds were screened for their cytotoxic activity using 60 cancer cell lines according to NCI (USA) protocol. Compound 19 showe

Full text of DOI:10.1016/j.ejmech.2018.07.004

Accepted Manuscript
Synthesis and anticancer activity of new dihydropyrimidinone derivatives
Amany S. Mostafa, Khalid B. Selim
PII:
S0223-5234(18)30560-9
10.1016/j.ejmech.2018.07.004
EJMECH 10539
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 April 2018
Revised Date: 1 July 2018
Accepted Date: 2 July 2018
Please cite this article as: A.S. Mostafa, K.B. Selim, Synthesis and anticancer activity of new
dihydropyrimidinone derivatives, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.07.004.
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Graphical Abstract
Synthesis and anticancer activity of new dihydropyrimidinone derivatives
Amany S. Mostafa and Khalid B. Selim
A series of dihydropyrimidinone derivatives bearing various heteroaryl moieties was
designed, synthesized and evaluated for anticancer activity. Compound 19 possessed
the most significant activity against NCI-H460, SK-MEL-5 and HL-60(TB) cell lines.
It proved to have dual inhibitory effect against VEGFR-2 and m TOR. Cell cycle
analysis of A549 cells showed cell cycle arrest at G2/M phase and pro-apoptotic
activity.

ACCEPTED MANUSCRIPT
Synthesis and anticancer activity of new dihydropyrimidinone derivatives
Amany S. Mostafa and Khalid B. Selim*
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt
*Corresponding author: K. B. Selimꢀ
e-mail: khbselim@mans.edu.eg
tel: +2050-2247496 fax: +2050-2247496
Abstract
A series of dihydropyrimidinone derivatives bearing various N-heterocyclic moieties was designed and
synthesized. Twelve new compounds were screened for their cytotoxic activity using 60 cancer cell lines
according to NCI (USA) protocol. Compound 19 showed a significant activity against NCI-H460, SK-
MEL-5, and HL-60(TB) cell lines with growth inhibition 88%, 86% and 85%, respectively, and was
found to be more safe on normal cells when compared to doxorubicin. Enzyme inhibition assay was
performed for compound 19 against mTOR (IC50 = 0.64 µM) and VEGFR-2 (IC50 = 1.97 µM) to show
high potency in comparison to rapamycin (IC50 = 0.43 µM) and sorafenib (IC50 = 0.3 µM) as references,
respectively. Cell cycle analysis of A549 cells treated with 19 showed cell cycle arrest at G2/M phase
and pro-apoptotic activity as indicated by annexin V-FITC staining.
Keywords Dihydropyrimidinone; anticancer; mTOR; VEGFR-2; cell cycle analysis; apoptosis
1. Introduction
Tyrosine kinases (TKs) are responsible for the phosphorylation of tyrosine residues in proteins leading
to change the function of protein and consequently involve in cell signaling pathways [1]. Mutations can
affect some TKs to become constantly active, leading to the outgrowth of cancer [2]. Among these TKs,
vascular endothelial growth factor (VEGF) is an important signaling protein engaged in both
vasculogenesis and angiogenesis. VEGF exerts its biological effects through binding to specific TK
receptors including VEGFR-1 and VEGFR-2 (Figure 1) [3,4]. It also acts as a survival or an anti-
apoptotic factor where it induces B-cell lymphoma 2 (Bcl-2) in endothelial cells as well as in breast
cancer cells [5,6]. Therefore, some therapeutic approaches using anti-VEGF/VEGFR2 are currently
explored to inhibit proliferation and induce apoptosis of malignant cells in hematologic diseases [7].
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase which plays a critical role in
phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, either through direct
phosphorylation or inhibition of the protein phosphatase 2 (PP2A) (Figure 1) [8,9]. This pathway is
important in regulating various cell functions, including cell growth, proliferation, survival, autophagy,
metabolism and cytoskeletal organization in many cancers. In addition, mTOR has profound effects on
the regulation of the programmed cell death, known as apoptosis, which has a significant function in
various physiological and pathophysiological circumstances [10,11,12]. mTORs are frequently
deregulated in diverse human cancers such as lung cancer, breast cancer and glioblastoma. Based on
their vital cellular functions, mTORs have emerged as effective anti-cancer drug targets.
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Figure 1. Brief role of VEGF and PI3K/AKT/mTOR pathway in cell growth.
Dihydropyrimidinone (DHPM) derivatives were discovered by Biginelli [13] through multi-component
reaction. DHPM is characterized by its multi-functionalized scaffold that exhibits diverse biological
activities [14], such as calcium channel blockade, anti-microbial [15], anti-viral [16], anti-oxidant
[17,18] and especially anti-cancer activity (Figure 2). These compounds were identified as promising
anticancer agents in which monastrol was the most highlighted [19]. Monastrol causes mitotic arrest at
G2/M phase by blocking bipolar mitotic spindle in mammalian cells leading to cell apoptosis [20].
Since then, monastrol has inspired the medicinal chemists to design new anticancer agents through
molecular manipulation of the DHPM scaffold by modifying the substituents regarding their electronic
and lipophilic nature. Extensive substitutions (R1, R2, R3, R4 and X) were placed on different positions
of the ring aiming to obtain potent anticancer agents such as dimethylenastron [21], compounds 1 [22], 2
[23] and 3 [24] (Figure 2). However, modification of substituent R5 at C6 has not been well exploited,
where most of the reports were limited to compounds that show antihypertensive activity other than
anticancer activity.
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Figure 2. Structures of some DHPM derivatives with anticancer activity.
On the other hand, the azole heterocycles (including thiazoline, tetrazole, 1,3,4-oxadiazole,
benzimidazole, 1,2,3-triazole, and cyclic amines) play an important role in improving pharmacokinetic
and pharmacodynamic properties of anticancer drugs by enhancing lipophilicity, polarity or the
physicochemical properties (Figure 3) [25, 26]. The presence of dithiocarbamate linkage in various
natural and synthetic molecules was proved to be crucial to their inhibitory activities against cancer cells
[27]. These findings have encouraged us to investigate the potential synergistic effect of the molecular
hybridization between DHPM nucleus and azole heterocycles via a methyl (thio) or dithiocarbamate
bridge at C6 with the aim of exploring the impact of such modification on the anticancer activity.
Figure 3. Molecular hybridization of DHPM scaffold with different azole heterocyles at C6.
Taking into consideration the value of DHPM scaffolds, this work aimed to construct novel candidates
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of anticancer drugs structurally related to monastrol, using the Biginelli reaction. Twelve of the
synthesized compounds were evaluated for their in vitro cytotoxicity according to the current single-
dose protocol of the National Cancer Institute (NCI) assay against a panel of 60 tumor cell lines. The
most potent compounds were further investigated for their activities against human normal cell lines
(MCF5, BJ, WPE1-NA22) and for their effect on cell death and cell cycle distribution as well as the
enzymatic inhibitory activity on mTOR and VEGFR (as a possible credible mechanism behind their
anticancer efficacy).
2. Results and discussion
2.1. Chemistry
Scheme 1. Synthesis of intermediate 5. Reagents and conditions: (a) HCl, EtOH, reflux, 3 h, 60%; (b)
Br2, CHCl3, rt, 12 h, 70%.
As shown in scheme 1, the starting DHPM 4 was conveniently prepared via multicomponent reaction, of
Biginelli type. It involved the cyclocondensation of benzaldehyde, N-methyl urea and ethyl acetoacetate
in presence of concentrated hydrochloric acid [28]. The intermediate 6-bromomethyl-DHPM 5 was
synthesized by treating with Br2 in CHCl3 [29].
Scheme 2. Synthesis of hybrids 6–9. Reagents and conditions: (a) RR'NH, DMF, K2CO3, rt, 8 h; (b)
NaN3, MeOH, rt, 48 h; (c) Propargyl alcohol, Na-ascorbate, CuSO4.5 H2O, DMF, rt, 36 h.
Direct amination of intermediate 5 with pyrrolidine and dicyclohexylamine gave hybrids 6 and 7,
respectively (Scheme 2). While, DHPM-1,2,3-triazole hybrid 9 was prepared via click chemistry
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approach through Cu(I)-catalyzed [3+2] cycloaddition of the known 6-azido-methyl-
dihydropyrimidinone 8 [30,31] with propargyl alcohol. The reported CuSO4/sodium ascorbate
conditions was utilized to obtain 9 as a single 1,4-disubstituted regioisomer [32,33].
Scheme 3. Synthesis of hybrids 10–16. Reagents and conditions: (a) RSH, DMF, K2CO3, rt, 6 h.
A new series of 5-membered heterocycles hybridized with DHPM nucleus at C6 through methyl sulfide
bridge 10–16 was prepared by direct nucleophilic substitution under basic condition between bromo
compound 5 and a wide variety of thiol derivatives (Scheme 3).
Scheme 4. Synthesis of hybrids 17–22. Reagents and conditions: (a) RR'NH, CS2, DMF, Na3PO4, rt, 4 h.
A series of dithiocarbamate-DHPM derivatives 17–22 was synthesized and their structures are shown in
scheme 4. The spectroscopic data (1H and 13C-NMR) of the newly synthesized compounds are in
agreement with the expected structures as described in the experimental section.
2.2. Biological evaluation
2.2.1. In vitro one-dose anticancer screening
Out of the newly synthesized sixteen target compounds, twelve hybrids namely 6, 7, 9–12, 15, 17–20
and 22 (Table 1); were submitted to Developmental Therapeutic Program-National Cancer Institute,
Bethesda, USA (www.dtp.nci.nih.gov) to be evaluated for their in vitro anticancer activity initially at a
single dose (10 µM) towards a panel of NCI 60 cancer cell lines including leukemia, melanoma, lung,
colon, CNS, ovarian, renal, prostate and breast cancers. The data are reported as a mean-graph of the
percent growth of treated cells compared to the untreated control cells, and presented as percentage
growth inhibition (GI %) caused by the test compounds (Table 1).
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Data analysis of growth inhibition assay showed that, compounds 19 and 22 have broad spectrum
cytotoxic activity with various degrees of growth inhibition against the tested tumor cell lines.
Compound 17 showed a potent activity against leukemia cell line K-562 with GI% of 83. Compounds
19 and 20 and 22 have moderate activity against leukemia SR cell line; colon cancer HCT-116, HCT-15
and prostate cancer PC-3 with GI% range of 41-79. Moreover, compound 7 showed moderate activity
against prostate cancer PC-3 and breast cancer T-47D with GI% values of 58 and 46, respectively.
Compound 10 showed moderate activity against leukemia SR with GI% value of 40.
Table 1. Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM
concentration of the selected compounds.
% Growth Inhibition (GI%)
Subpanel cancer
cell Lines
6
7
9
10
11
12
15
17
18
19
20
22
Leukemia
K-562
MOLT-4
SR
1
4
10
-
31 10
35 11
24
19
40
10
17
26
13
7
10
14
1
34
29
16
11
20
30
27
33
22
24
44
20
83
5
12
4
65
72
71
85
47
49
63
32
20
2
HL-60(TB)
37
2
6
Non-Small Cell Lung Cancer
A549/ATCC
NCI-H322M
NCI-H522
-
9
4
-
33
5
18
-
9
5
-
16
27
19
2
-
-
12
3
5
15
16
-
4
18
7
29
15
25
10
27
15
28
12
33
17
37
39
53
9
42
18
14
-
NCI-H460
13
-
-
88
Colon Cancer
HCT-116
2
6
33
11
5
6
10
28
12
8
6
7
19
15
6
7
34
35
74
61
41
48
57
60
HCT-15
CNS Cancer
SF-295
9
19
4
10
11
7
14
9
13
17
15
61
Melanoma
LOX IMVI
UACC-62
SK-MEL-5
Ovarian Cancer
OVCAR-4
8
-
-
13
23
19
7
-
-
16
27
4
14
17
2
11
11
-
27
10
10
12
16
1
11
28
25
13
24
23
33
42
51
67
54
86
-
3
26
7
3
4
16
15
4
14
-
6
14
15
-
6
16
12
15
5
29
13
56
37
NCI/ADR-RES
Renal Cancer
ACHN
CAKI-1
UO-31
-
9
8
13
30
27
2
8
7
17
24
21
3
14
21
2
2
4
3
14
34
7
-
9
15
17
29
11
15
37
21
26
55
63
75
56
Prostate Cancer
PC-3
-
-
-
-
8
20
-
27
58
79
41
53
Breast Cancer
MCF-7
T-47D
MDA-MB-468
13
1
-
36 12
19
37
11
15
17
3
14
-
1
11
25
17
14
18
6
12
45
20
15
37
14
30
59
21
54
50
53
1
-
46
21
The data showed that compounds bearing different heterocyclic moieties and linked to either -CH2- (6,
7, 9) or -CH2S- (e.g. 10–12 and 15) have week activity towards the tested cell lines. While compounds
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bearing dithiocarbamate linkage (e.g. 17–20 and 22) exhibited moderate to high inhibitory activity.
Among DHMP-dithiocarbamate hybrids, compound 19 with dicyclohexylamino group exhibited potent
and broad activity against several types of cancer cell lines rather than compounds with morpholine,
piperidine, or phenyl piperazine moieties. Compound 19 showed a significant anticancer activity against
leukemia HL-60(TB), non-small cell lung cancer NCI-H460, and melanoma SK-MEL-5 with GI% 85,
88, and 86, respectively Removing the dithiocarbamate group from compound 19 gives compound 7
.
and led to loss of its functional activity.
Figures 4A and 4B show the effect of dithiocarbamate linkage on the functional activity of 19 and 7
towards the 60 NCI cell lines.
100
Compound 7
Compound 19
80
60
40
20
0
Figure 4A. A comparison between 19 (with dithiocarbamate) and 7 (without dithiocarbamate) (GI %) of
in vitro subpanel tumor cell lines. Leukemia: CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR. Non-
Small Cell Lung Cancer: A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-
H522. Colon Cancer: COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620. CNS Cancer: SF-268, SF-295,
SF-539, SNB-19, SNB-75, U251. Prostate Cancer: PC-3, DU-145.
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100
80
60
40
20
0
Compound 7
Compound 19
Figure 4B. A comparison between 19 (with dithiocarbamate) and
7
(without dithiocarbamate) (GI %) of
in vitro subpanel tumor cell lines. Melanoma: LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-
MEL-28, SK-MEL-5, UACC-257, UACC-62. Ovarian Cancer: IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8,
NCI/ADR-RES, SK-OV-3. Renal Cancer: 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31. Breast
Cancer: MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468.
The IC50 of compounds 13, 14, 16 and 21 (that were not selected by NCI) were determined to assess
their cytotoxic activity against prostate cancer PC-3, colon cancer HCT-116 and breast cancer MCF-7
(Table 2). The results indicate that compound 14 with nitro group at position 5 is the most active
member among benzimidazole thioether series with high cytotoxic activity (IC50 = 9.18, 7.29 and 9.39
µM) against PC-3, HCT-116 and MCF-7, respectively. While compound 16 with p-tolyl substituent on
the oxadiazole ring has moderate activity (IC50 = 36.04 and 25.26 µM) against HCT-116 and MCF-7,
respectively. Moreover, the IC50 data shows that compound 21 exhibits weak inhibitory activities
(ranging from 53.75 to 72.29 µM) against the three cell lines. The results were compared with
doxorubicin (DOX) as a reference drug.
Table 2. Cytotoxic activity (IC50) of some hybrids against PC-3, HCT-116 and MCF-7 cells.
In vitro Cytotoxicity IC50 (µM)
Compounds
PC-3
HCT-116
9.50 ± 0.8
MCF-7
22.29 ± 2.1
19.36 ± 1.8
13
9.18 ± 0.9
61.83 ± 3.8
72.29 ± 4.4
8.87 ± 0.6
7.29 ± 0.6
36.04 ± 2.5
67.60 ± 3.9
5.23 ± 0.3
9.39 ± 1.1
25.26 ± 2.3
53.75 ± 3.5
4.17 ± 0.2
14
16
21
DOX
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Data are shown as means ± SD of three independent experiments.
2.2.2. In vitro cytotoxicity against human normal cells
To investigate whether the synthesized hybrids showed selective cytotoxicity between normal and
cancer cells, the most active hybrids 14 and 19 were evaluated against three human normal cell lines
including MRC5 (lung), BJ (skin) and WPE1-NA22 (prostate). As shown in figure 5, compounds 14 and
19 exhibited lower toxicity towards MRC5 (IC50 = 44.16 and 32.04 µM) in comparison to DOX ((IC50 =
14.76 µM). Compound 14 had a lower toxic effect than compound 19 and DOX on the normal BJ. On
the other hand, compound 19 induce lower toxicity (IC50 = 32.09 µM) on WPE1-NA22 cells as
compared with compound 14 and DOX (Table 3).
Figure 5. Cytotoxic effect of compounds 14 and 19 in comparison to doxorubicin on lung MRC5 (A),
skin BJ (B) and prostate WPE1-NA22 (C) cell lines. Data are shown as means ± SD of three
experiments. Data were fitted using linear regression.
Table 3. Cytotoxic activity (IC50) of 14 and 19 hybrids against human normal cells.
In vitro Cytotoxicity IC50 (µM)
Compounds
MRC5
BJ
WPE1-NA22
14
19
DOX
44.16 ± 1.93
32.04 ± 1.0
14.76 ± 0.47
37.47 ± 3.43
18.18 ± 1.06
17.62 ± 1.01
21.73 ± 1.54
32.09 ± 2.74
18.25 ± 2.43
Data are shown as means ± SD of three independent experiments.
2.2.3. Effects on the levels of active caspase-3 and caspase-9
Caspases, cysteine-containing aspartic acid-specific proteases, provide pivotal links in cell regulatory
networks controlling cell death. Caspase-3 is a key executioner protease which is activated by upstream
initiator caspases as caspase-9 [34,35].
Herein, the study was further extended to investigate the ability of compound 19 to provoke apoptosis in
A549 lung cell line. Treatment of A549 cell line with compound 19 significantly increased the
expression levels of active caspases-3 and caspases-9 by about 10 and 100 folds, respectively, in
comparison to the control, 5-fluorouracil (5-FU) (Table 4).
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Table 4. Effect of hybrid 19 on caspase-3 and caspase-9 enzyme assay in A549 cells.
Compound Conc. (µM)
Caspase-3 ng/mL
Caspase-9 ng/mL
Control
5-FU
19
-
53.45
582.1
560.4
0.1726
16.84
18.14
7.4
3.04
2.2.4. Effect on cell cycle progression
To gain further evidence regarding the role of the synthesized hybrids in growth inhibition of cancer
cells, measuring of cell cycle arrest and induction of apoptosis was performed in different phases. Thus,
we started to investigate the effect of compound 19 on cell cycle distribution in A549 cells. The cells
were treated with compound 19 at a concentration of 2 µM for 24 h, stained with propidium iodide (PI),
and analyzed by flow cytometer (FCM) according to the literature. Normal lymphocyte DNA was used
to set diploid G0-G1 phase and the untreated cells exhibited significant arrest in synthetic S-phase. The
results showed that cells were arrested in G2-M phase of the cell cycle at 24 h by 12.4% (Table 5).
Additionally, the hybrid 19 at 24 h induced block in G0-G1 phase by 58.3% as compared to that of
positive control 5-FU (47.4%). The induced apoptosis was determined by measuring the percent of cells
stalled in the pre-G1 peak: 7.4 and 15.5% of cells were found in the pre-G1 peak after 48 h exposure of
19 and 5-FU, respectively compared to the untreated A549 cells.
Table 5. Effect of hybrid 19 on the cell cycle progression in A549 cells using flow cytometry.
Cell cycle distribution (%)
Compound
G0/G1
S
G2/M
Pre/G1
0.92
Control
5-FU
19
67.62
25.26
6.2
47.36
58.25
16.08
21.95
21.04
12.41
15.52
7.39
2.2.5. Detection of apoptosis
Extrinsic and intrinsic apoptosis in A549 cells induced by hybrid 19 were also evaluated by Annexin V
and PI staining. In this study A549 cells were incubated with 19 at 2 µM concentration for 24 h.
Compound 19 was found to induce early apoptosis (3.27%) in A549 at 24 h and enhanced late apoptotic
induction (3.31%) by 10 folds over the control. Cumulatively, 19 induced apoptosis with 6.6%, and the
results were compared with 5-FU (Figure 6B). This finding is consistent with the data obtained from cell
cycle analysis in figure 6A.
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A
19
5-FU
Control
B
19
5-FU
Control
Figure 6. A) Effect of compound 19 on the cell cycle distribution of A549 cell line. B) Apoptosis effect
on A549 cell line induced by compound 19.
2.2.6. VEGFR-2 and mTOR enzyme assay inhibition
The most active anticancer agents (14 and 19) were screened as inhibitors of VEGFR-2 and mTOR and
the IC50 results are shown in table 6. Compounds 14 and 19 showed high inhibitory activity with IC50
values of 1.20
respectively in comparison to the reference drug, sorafinib, with IC50 of 0.32
inhibition (Table 6). In addition, both compounds showed high inhibitory activity against mTOR with
IC50 values of 0.72 M for 14 and 0.64 M for 19 with % inhibition of 84.58% and 86.57%, respectively
in comparison to the reference drug, rapamycin, with IC50 of 0.43 M, and 94.88% mTOR inhibition
µM for 14 and 1.97
µ
M for 19 with 75.99% and 68.52% VEGFR-2 inhibition,
µM, and 91.49% VEGFR-2
µ
µ
µ
(Table 6). These results indicate that DHPM derivatives contribute more to the interaction with mTOR
enzyme rather than VEGFR-2 enzyme.
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Table 6. Inhibitory activities and cytotoxicity of 14 and 19 (IC50 µM) against VEGFR-2 and mTOR
enzymes against Hela cell line.
VEGFR-2
mTOR
IC50 (µM)
Hela cells
Compound
% inhibition IC50 (µM) % inhibition
IC50 (µM)
0.72
0.64
-
1.40±0.08
1.85±0.1
75.99
68.52
91.49
-
1.2
1.97
0.32
-
84.58
86.57
-
14
19
Sorafinib
Rapamycin
0.64±0.05
0.77±0.08
94.88
0.43
2.2.7. Physicochemical properties and Lipinski’s rule of five
The most active synthesized compound 19 was evaluated to test its compliance to the Lipinski’s rule of
five in comparison to rapamycin. Calculations were performed by SwissADME web service [36].
Molecules violating more than one of these rules may have problems with bioavailability. Predictions of
ADME properties for the studied compound 19 are given in table 7. The results showed that compound
19 complies with these rules, while rapamycin does not, suggesting that compound 19 would possess
drug-like characters. Predictions of water solubility of 19 and rapamycin revealed that both compounds
have poor solubility, however, the topological polar surface area (TPSA) value for 19 is 119.27 in
comparison to rapamycin (TPSA = 195.43) and theoretically it may confer better passive oral absorption
for 19 than rapamycin.
Table 7. Solubility, topological surface area and calculated Lipinski’s rule of five for compound 19 and
rapamycin.
Compound Log Sa TPSAb
MWc
Mlog Pd nHBAe nHBDf nRBg nVioh
-6.15
19
Rapamycin -8.90
119.27
195.43
529.76 3.72
914.17 1.00
3
1
3
10
6
1
2
13
aSolubility parameter. bTopological polar surface area (Ų). cMolecular weight. dLipophilicity parameter. eNumber
f
g
h
of hydrogen bond acceptors. Number of hydrogen bond donors. Number of rotatable bonds. Number of
violations to Lipinski’s rule of five.
3. Conclusion
Several hybrids of DHPM with N-hterocyclic moieties were synthsized and evaluated for their
anticancer activity against a panel of cancer cell lines according to NCI (USA) protocol. DHPM with
dithiocarbamate moiety 19 was identified as the most promising anticancer candidate against NCI-H460,
SK-MEL-5, and HL-60(TB). The inhibitory activities against VEGFR-2 and mTOR as well as the
induction of apoptosis of 19 were also investigated.
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4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured using Fischer-John device and are uncorrected. IR spectra were recorded
1
on Nicolet IS 10 FT-IR spectrometer in KBr discs. The H NMR spectra were recorded on Bruker 200
MHz (for 6 and 10), Bruker 400 MHz (for 11–19, 22) or Jeol 500 MHz (for 7, 9, 20, 21) NMR
spectrometer, while the 13C NMR spectra were recorded on Bruker 100 MHz (for 6, 10–19, 22) or Jeol
125 MHz (for 7, 9, 20, 21) NMR spectrometer, with the deuterated solvent as the lock and residual
solvent as the internal refrence. All chemical shift values, coupling constants J and the multiplicity (s =
singlet, d = doublet, t = triplet, m = multiplet, br = broad) are quoted in ppm and in Hz, respectively. The
intermediates 4 [28], 5 [29] and 8 [30,31] were prepared according to the described method. All
chemicals and reagents were commercially availble from Aldrich, Fluca or Merck.
4.1.2. Ethyl 1,6-dimethyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4)
To a mixture of benzaldehyde (10 g, 2 mmol), ethyl acetoacetate (12.26 g, 2.4 mmol) and N-methyl urea
(8.38 g, 2.4 mmol) in EtOH (40 mL), concentrated hydrochloric acid (0.5 mL) was added. The mixture
was stirred under reflux for 3 h. After cooling the solution to 0 °C for 1 h, the formed precipitate was
collected, dried and recrystallized from EtOH to give compound 4 (15.6 g, 60%) with m.p. 176–177 °C
(lit. 175–176 ºC) [28].
4.1.3. Ethyl 6-(bromomethyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (5)
A solution of Bromine (1.6 g, 0.01 mol) in CHCl3 (50 mL) was slowly added to a stirred solution of 4
(2.47 g, 0.01 mol) in CHCl3 (30 mL) at 4 °C over 6 h. The reaction mixture was warmed to rt, stirred for
12 h, and the solvent was removed under reduced pressure. The formed precipitate was crystallized from
EtOH to give compound 5 (2.22 g, 70%) with m.p. 170–171 °C (lit. 170 °C) [29].
4.1.4. General procedure for preparation compounds
6 and 7.
To a solution of compound 5 (0.35 g, 1 mmol) in DMF (5 mL), the appropriate amine (1 mmol) and
K2CO3 (0.14 g, 1 mmol) was added and the mixture was stirred for 8 h at room temperature. The
reaction mixture was poured into a mixture of ice and water and stirred for 10 min. The separated solid
was filtered, washed with cold water and recrystallized from EtOH to afford pure product of compounds
6 and 7.
4.1.4.1.
carboxylate (
Yield (0.23 g, 67%) as white solid with m.p. 169–170 °C. IR (KBr) (
Ethyl
1-methyl-2-oxo-4-phenyl-6-(pyrrolidin-1-ylmethyl)-1,2,3,4-tetrahydropyrimidine-5-
6
)
υ
, cm-1): 1508 (C=C str.), 1637
1
(C=O amide), 1725 (C=O ester), 2977 (C-H Ar. str.), 3241 (NH). H-NMR (200 MHz, DMSO): 1.16
(3H, t, J = 7.1 Hz, CH3-CH2), 1.65-1.71 (4H, m, pyrrolidine-2CH2), 2.52 (4H, dd, J = 3.6, 1.8 Hz,
pyrrolidine-2CH2), 3.25 (3H, s, N-CH3), 3.89 (1H, d, J = 13.2 Hz, CH2-N), 4.01 (1H, d, J = 13.2 Hz,
CH2-N), 4.08 (2H, q, J = 7.1 Hz, CH2-CH3), 5.20 (1H, d, J = 3.6 Hz, pyrimidine-CH), 7.23 (1H, m, Ar-
H), 7.27-7.30 (2H, m, Ar-H), 7.31-7.40 (2H, m, Ar-H), 7.98 (1H, d, J = 3.6 Hz, NH). 13C-NMR (100
MHz, DMSO): 14.0, 24.3, 29.6, 52.3, 55.1, 57.6, 60.2, 105.3, 126.4, 127.6, 128.6, 143.5, 147.8, 153.1,
165.6. EI-MS: m/z (%): 343 (M+, 72). Anal. Calcd. for C19H25N3O3 (343.19): C, 66.45; H, 7.34; N,
12.24. Found: C, 66.42; H, 7.35; N, 12.23.
13

ACCEPTED MANUSCRIPT
4.1.4.2. Ethyl 6-((dicyclohexylamino)methyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (7)
Yield (0.24 g, 54%) as white solid with m.p. 187–188 °C. 1H-NMR (500 MHz, DMSO): 0.94-1.11 (6H,
m, cyclohexyl-H), 1.14 (3H, t, J = 6.9 Hz, CH3-CH2), 1.22-1.39 (5H, m, cyclohexyl-H), 1.47-1.55 (2H,
m, cyclohexyl-H), 1.55-1.64 (5H, m, cyclohexyl-H), 1.64-1.1.75 (4H, m, cyclohexyl-H), 3.27 (3H, s, N-
CH3), 3.88 (1H, d, J = 13.4 Hz CH2-N), 4.07 (2H, q, J = 6.9 Hz, CH2-CH3), 4.21 (1H, d, J = 13.4 Hz
CH2-N), 5.19 (1H, d, J = 2.8 Hz, pyrimidine-CH), 7.21 (2H, d, J = 7.1 Hz, Ar-H), 7.25 (1H, m, Ar-H),
7.32 (2H, t, J = 7.1 Hz, Ar-H), 7.91 (1H, d, J = 2.8 Hz, NH). 13C-NMR (125 MHz, DMSO): 13.9, 24.7,
25.8, 29.6, 30.1, 45.5, 52.4, 60.2, 62.3, 106.1, 126.2, 127.2, 128.8, 143.3, 147.7, 153.1, 164.9. EI-MS:
m/z (%): 453(M+, 66). Anal. Calcd. for C27H39N3O3 (453.30): C, 71.49; H, 8.67; N, 9.26. Found: C,
71.45; H, 8.70; N, 9.24.
4.1.4.3 Ethyl 6-(azidomethyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(8)[30,31]
Sodium azide (0.1 g, 1.5 mmol) was added to a solution of compound 5 (0.35 g, 1 mmol) in MeOH (10
mL) and the mixture was stirred at room temperature for 48 h. After completion of the reaction, the
solvent was evaporated under reduced pressure and the produced product was washed with water, dried
to afford white solid (0.23 g, 75 % yield) and used without further purification in the next step.
4.1.6. Ethyl 6-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (9)
To a solution of azide 8 (0.32 g, 1 mmol) in DMF (5 mL), propargyl alcohol (0.06 g, 1 mmol), CuSO4.5
H2O (5 mg, 2 mol%), and sodium ascorbate (40 mg, 0.2 mmol) were added and the mixture was stirred
at room temperature for 36 h. The reaction mixture was poured into a mixture of ice and water and
stirred for 2 h. The separated solid was collected by filtration, washed with water, dried and
recrystallized from a mixture of EtOH and water to afford compound 9 (0.27 g, 72% yield) as beige
solid with m.p. 172-173 °C. IR (KBr) (
υ
, cm-1): 1498 (C=C str.), 1644 (C=O amide), 1723 (C=O ester),
1
2968 (C-H Ar. str.), 3211 (O-H str.), 3257 (NH). H-NMR (500 MHz, DMSO): 1.11 (3H, t, J = 7.1 Hz,
CH3-CH2), 3.08 (3H, s, N-CH3), 4.08 (2H, q, J = 7.1 Hz, CH2-CH3), 4.52 (2H, d, J = 5.6 Hz, CH2OH),
5.20 (1H, t, J = 5.6 Hz, OH), 5.24 (1H, d, J = 3.8 Hz, pyrimidine-CH), 5.81 (1H, d, J = 15.0 Hz, CH2-
N), 6.00 (1H, d, , J = 15.0 Hz, CH2-N), 7.26-7.31 (3H, m, Ar-H), 7.33-7.36 (2H, m, Ar-H), 7.93 (1H, s,
13
triazole-CH), 8.14 (1H, d, J = 3.8 Hz, NH). C-NMR (125 MHz, DMSO): 14.3, 30.0, 46.6, 53.0, 55.5,
60.9, 105.4, 123.1, 126.7, 128.2, 129.1, 143.5, 145.6, 148.8, 153.4, 165.4. EI-MS: m/z (%): 371 (M+,
34). Anal. Calcd. for C18H21N5O4 (371.16): C, 58.21; H, 5.70; N, 18.86. Found: C, 58.22; H, 5.75; N,
18.88.
4.1.5. General procedure for preparation compounds 10–16.
To a solution of compound 5 (0.35 g, 1 mmol) in DMF (5 mL) the appropriate thiol (1 mmol) and
K2CO3 (0.14 g, 1 mmol) were added and the mixture was stirred for 6 h at room temperature. The
reaction mixture was poured into crushed ice and stirred for 1 h. The separated solid was filtered,
washed with ice-cold water and recrystallized from EtOH to afford compounds 10–16.
4.1.7.1. Ethyl 6-((4,5-dihydrothiazol-2-ylthio)methyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (10
)
Yield (0.24 g, 62%) as white solid with m.p. 150–151 °C. 1H-NMR (200 MHz, DMSO): 1.13 (3H, t, J =
7.1 Hz, CH3-CH2), 3.16 (3H, s, N-CH3), 3.49 (2H, t, J = 8.0 Hz, thiazole-CH2), 4.06 (2H, q, J = 7.1 Hz,
CH2-CH3), 4.16 (2H, t, J = 8.0 Hz, thiazole-CH2), 4.67 (1H, d, J = 13.1 Hz, S-CH2), 4.79 (1H, d, J =
13.1 Hz, S-CH2), 5.14 (1H, d, J = 3.8 Hz, pyrimidine-CH), 7.19-7.23 (2H, m, Ar-H), 7.26 (1H, m, Ar-
14

ACCEPTED MANUSCRIPT
13
H), 7.31-7.36 (2H, m, Ar-H), 8.07 (1H, d, J = 3.8 Hz, NH). C-NMR (100 MHz, DMSO): 13.9, 29.6,
30.3, 35.5, 52.3, 59.6, 60.2, 105.9, 126.1, 127.6, 128.7, 143.2, 147.6, 153.1, 163.9, 166.0. EI-MS: m/z
(%): 391 (M+, 23). Anal. Calcd. for C18H21N3O3S2 (391.10): C, 55.22; H, 5.41; N, 10.73. Found: C,
55.25; H, 5.38; N, 10.77.
4.1.5.1. Ethyl 1-methyl-2-oxo-4-phenyl-6-((1-phenyl-1H-tetrazol-5-ylthio)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (11
)
Yield (0.23 g, 52%) as white solid with m.p. 109–110 °C. 1H-NMR (400 MHz, CDCl3): 1.20 (3H, t, J =
7.1 Hz, CH3-CH2), 3.41 (3H, s, N-CH3), 4.15 (2H, q, J = 7.1 Hz, CH2-CH3), 4.94 (1H, d, J = 12.8 Hz,
CH2-S), 5.15 (1H, d, J = 12.8 Hz, CH2-S), 5.39 (1H, s, Pyrimidine-CH), 5.79 (1H, s, NH), 7.24-7.29
(2H, m, Ar-H), 7.30-7.34 (3H, m, Ar-H), 7.59 (5H, s, Ar-H). 13C-NMR (100 MHz, DMSO): 13.9, 29.6,
30.3, 52.3, 60.2, 105.8, 124.1, 126.4, 127.6, 128.5, 128.9, 129.0, 134.2, 143.3, 147.6, 153.1, 156.7,
166.0. EI-MS: m/z (%): 450 (M+, 68). Anal. Calcd. for C22H22N6O3S (450.15): C, 58.65; H, 4.92; N,
18.65. Found: C, 58.61; H, 4.88; N, 18.63.
4.1.5.2. Ethyl 6-((1H-benzo[d]imidazol-2-ylthio)methyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (12
)
Yield (0.27 g, 65%) as white solid with m.p. 134–135 °C. 1H-NMR (400 MHz, DMSO): 1.10 (3H, t, J =
7.1 Hz, CH3-CH2), 3.21 (3H, s, N-CH3), 4.03 (2H, q, J = 7.1 Hz, CH2-CH3), 4.81 (1H, d, J = 13.0 Hz,
CH2-S), 5.00 (1H, d, J = 13.0 Hz, CH2-S), 5.15 (1H, d, J = 3.8 Hz, pyrimidine-CH), 7.12-7.16 (2H, m,
Ar-H), 7.21-7.29 (3H, m, Ar-H), 7.31-7.35 (2H, m, Ar-H), 7.39 (1H, d, J = 7.0 Hz, Ar-H), 7.53 (1H, d, J
= 6.3 Hz, Ar-H), 8.07 (1H, d, J = 3.8 Hz, pyrimidine-NH), 12.68 (1H, s, benzimidazole-NH). 13C-NMR
(100 MHz, DMSO): 13.9, 29.6, 29.7, 52.4, 60.2, 105.4, 110.6, 117.6, 121.3, 122.0, 126.1, 127.6, 128.5,
128.6, 135.5, 143.3, 143.5, 147.8, 153.1, 164.9. EI-MS: m/z (%): 422 (M+, 15). Anal. Calcd. for
C22H22N4O3S (422.14): C, 62.54; H, 5.25; N, 13.26. Found: C, 62.52; H, 5.26; N, 13.25.
4.1.5.3. Ethyl 1-methyl-6-((5-methyl-1H-benzo[d]imidazol-2-ylthio)methyl)-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (13
Yield (0.26 g, 61%) as white solid with m.p. 98–99 °C. IR (KBr) (
amide), 1721 (C=O ester), 3021 (C-H Ar. str.), 3248 (NH), 3315 (NH). H-NMR (400 MHz, CDCl3):
1.14 (3H, t, J = 7.1 Hz, CH3-CH2), 2.44 (3H, s, benzimidazole-CH3), 3.35 (3H, s, N-CH3), 4.06 (2H, q, J
= 7.1 Hz, CH2-CH3), 4.79 (2H, s, CH2-S), 5.34 (1H, d, J = 3.0 Hz, pyrimidine-CH), 5.71 (1H, d, J = 3.0
Hz, pyrimidine-NH), 7.03 (1H, dd, J = 6.7, 6.7 Hz, Ar-H), 7,09 (1H, s, Ar-H), 7.16-7.22 (2H, m, Ar-H),
)
υ
, cm-1): 1487 (C=C str.), 1640 (C=O
1
13
7.23-6.26 (3H, m, Ar-H), 7.53 (1H, m, Ar-H), 10.25 (1H, s, benzimidazole-NH). C-NMR (100 MHz,
DMSO): 13.7, 21.3, 29.6, 30.2, 52.4, 60.2, 105.6, 111.0, 115.4, 121.3, 126.4, 127.3, 128.2, 128.6, 132.6,
135.9, 143.0, 143.5, 147.8, 153.1, 165.0. EI-MS: m/z (%): 436 (M+, 81). Anal. Calcd. for C23H24N4O3S
(436.16): C, 63.28; H, 5.54; N, 12.83. Found: C, 63.26; H, 5.58; N, 12.82.
4.1.5.4. Ethyl 1-methyl-6-((5-nitro-1H-benzo[d]imidazol-2-ylthio)methyl)-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (14
)
Yield (0.31 g, 67%) as light brown solid with m.p. 121–122 °C. 1H-NMR (400 MHz, CDCl3): 1.21 (3H,
t, J = 7.0 Hz, CH3-CH2), 3.41 (3H, s, N-CH3), 4.18 (2H, q, J = 7.0 Hz, CH2-CH3), 4.87 (1H, s, CH2-S),
4.98 (1H, s, CH2-S), 5.39 (1H, s, pyrimidine-CH), 5.94 (1H, s, pyrimidine-NH), 7.16-7.24 (2H, m, Ar-
H), 7.28-7.42 (3H, m, Ar-H), 7.44-7.60 (1H, m, Ar-H), 8.15 (1H, d, J = 8.4 Hz, Ar-H), 8.39 (1H, s, Ar-
H). 13C-NMR (100 MHz, DMSO): 13.8, 29.6, 29.9, 52.3, 60.2, 105.7, 111.2, 113.1, 117.2, 126.6, 127.6,
128.3, 128.5, 135.8, 143.4, 143.4, 143.5, 147.7, 153.1, 165.2. EI-MS: m/z (%): 467 (M+, 59). Anal.
Calcd. for C22H21N5O5S (467.13): C, 56.52; H, 4.53; N, 14.98. Found: C, 56.49; H, 4.56; N, 15.01.
15

ACCEPTED MANUSCRIPT
4.1.5.5. Ethyl 1-methyl-2-oxo-4-phenyl-6-((5-phenyl-1,3,4-oxadiazol-2-ylthio)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (15
)
Yield (0.3 g, 68%) as light brown solid with m.p. 79–80 °C. 1H-NMR (400 MHz, CDCl3): 1.19 (3H, t, J
= 7.1 Hz, CH3-CH2), 3.44 (3H, s, N-CH3), 4.13 (2H, q, J = 7.1 Hz, CH2-CH3), 4.85 (1H, d, J = 11.5 Hz,
CH2-S), 4.98 (1H, d, J = 11.5 Hz, CH2-S), 5.42 (1H, s, pyrimidine-CH), 5.67 (1H, s, NH), 7.26-7.39
(6H, m, Ar-H), 7.49-7.58 (2H, m, Ar-H), 7.89 (1H, d, J = 8.0 Hz, Ar-H), 8.01(1H, d, J = 7.1 Hz, Ar-H).
13C-NMR (100 MHz, DMSO): 13.7, 29.6, 30.3, 52.3, 60.2, 106.0, 120.3, 126.2, 126.3, 127.6, 128.1,
128.7, 142.7, 143.4, 147.8, 153.4, 161.5, 165.2, 166.6. EI-MS: m/z (%): 450 (M+, 100). Anal. Calcd. for
C23H22N4O4S (450.14): C, 61.32; H, 4.92; N, 12.44. Found: C, 61.30; H, 4.96; N, 12.43.
4.1.5.6. Ethyl 1-methyl-2-oxo-4-phenyl-6-((5-p-tolyl-1,3,4-oxadiazol-2-ylthio)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (16
)
Yield (0.32 g, 70%) as beige solid with m.p. 132–133 °C. 1H-NMR (400 MHz, CDCl3): 1.10 (3H, t, J =
7.1 Hz, CH3-CH2), 2.36 (3H, s, phenyl-CH3), 3.35 (3H, s, N-CH3), 4.03 (2H, q, J = 7.1 Hz, CH2-CH3),
4.74 (1H, d, J = 12.8 Hz, CH2-S), 4.88 (1H, d, J = 12.8 Hz, CH2-S), 5.32 (1H, s, pyrimidine-CH), 5.50
13
(1H, s, NH), 7.18-7.24 (7H, m, Ar-H), 7.80 (2H, d, J = 8.2 Hz, Ar-H). C-NMR (100 MHz, DMSO):
13.7, 21.2, 29.6, 30.3, 52.3, 60.2, 105.8, 120.2, 126.1, 126.4, 127.6, 128.6, 130.0, 142.4, 143.1, 147.6,
153.1, 161.3, 164.8, 166.2. EI-MS: m/z (%): 464 (M+, 47). Anal. Calcd. for C24H24N4O4S (464.15): C,
62.05; H, 5.21; N, 12.06. Found: C, 62.01; H, 5.23; N, 12.10.
4.1.6. General procedure for preparation compounds (17–22)
A mixture of the appropriate amine (1.5 mmol), carbon disulfide (0.45 mL, 7.5 mmol) and anhydrous
sodium phosphate (0.33 g, 2.0 mmol) in DMF (8 mL) was stirred at room temperature for 30 min.
Compound 5 (0.53 g, 1.5 mmol) was added, and stirred at room temperature for 4 h. The reaction
mixture was poured into a mixture of ice and water (60 mL). The separated solid was collected by
filtration, and recrystallized from a mixture of DMF and water to afford compounds 17–22.
4.1.6.1. Ethyl 1-methyl-6-((morpholine-4-carbonothioylthio)methyl)-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (17
Yield (0.37 g, 57%) as off-white solid with m.p. 120–121 °C. IR (KBr) (
)
υ
, cm-1): 850 (C-S str.), 1070
1
(C=S str.), 1512 (C=C str.), 1638 (C=O amide), 1720 (C=O ester), 2965 (C-H Ar. str.), 3244 (NH). H-
NMR (400 MHz, DMSO): 1.12 (3H, t, J = 7.1 Hz, CH3-CH2), 3.14 (3H, s, N-CH3), 3.67 (4H, s,
morpholine-2CH2), 3.93 (2H, s, morpholine-CH2), 4.05 (2H, q, J = 7.1 Hz, CH2-CH3), 4.22 (2H, s,
morpholine-CH2), 4.75 (1H, d, J = 12.5 Hz, CH2-S), 4.88 (1H, d, J = 12.5 Hz, CH2-S), 5.17 (1H, d, J =
3.6 Hz, pyrimidine-CH), 7.24 (2H, dd, J = 7.4, 7.4 Hz, Ar-H), 7.26-7.30 (1H, m, Ar-H), 7.34 (2H, dd, J
=7.4,7.4 Hz, Ar-H), 8.10 (1H, d, J = 3.6 Hz, NH). 13C-NMR (100 MHz, DMSO): 13.9, 29.6, 35.6, 51.5,
52.5, 60.2, 65.6, 106.1, 126.2, 127.6, 128.6, 143.3, 146.3, 152.9, 164.7, 193.7. EI-MS: m/z (%): 435
(M+, 55). Anal. Calcd. for C20H25N3O4S2 (435.13): C, 55.15; H, 5.79; N, 9.65. Found: C, 55.12; H, 5.78;
N, 9.65.
4.1.6.2. Ethyl 1-methyl-2-oxo-4-phenyl-6-((piperidine-1-carbonothioylthio)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (18
)
Yield (0.4 g, 62%) as white solid with m.p. 169–170 °C. 1H-NMR (400 MHz, DMSO): 1.12 (3H, t, J =
7.1 Hz, CH3-CH2), 1.53-1.63 (4H, m, piperidine-2CH2),1.63-1.70 (2H, m, piperidine-CH2), 3.13 (3H, s,
N-CH3), 3.88 (2H, br. s, piperidine-CH2), 4.05 (2H, q, J = 7.1 Hz, CH2-CH3), 4.23 (2H, br. s, piperidine-
CH2), 4.71 (1H, d, J = 12.5 Hz, CH2-S), 4.86 (1H, d, J = 12.5 Hz, CH2-S), 5.16 (1H, d, J = 3.8 Hz,
16

ACCEPTED MANUSCRIPT
pyrimidine-CH), 7.20-7.26 (2H, m, Ar-H), 7.27-7.29 (1H, m, Ar-H), 7.31-7.37 (2H, m, Ar-H), 8.09 (1H,
13
d, J = 3.8 Hz, NH). C-NMR (100 MHz, DMSO): 14.0, 23.7, 24.7, 29.6, 35.6, 50.9, 52.5, 60.2, 106.1,
126.2, 127.7, 129.0, 143.3, 146.4, 153.1, 165.0, 193.7. EI-MS: m/z (%): 433 (M+, 17). Anal. Calcd. for
C21H27N3O3S2 (433.15): C, 58.17; H, 6.28; N, 9.69. Found: C, 58.13; H, 6.26; N, 9.71.
4.1.6.3. Ethyl 6-((dicyclohexylcarbamothioylthio)methyl)-1-methyl-2-oxo-4-phenyl-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (19
Yield (0.38 g, 48 % yield) as off-white solid with m.p. 107–108 °C. IR (KBr) (
)
υ
, cm-1): 862 (C-S str.),
1084 (C=S str.), 1523 (C=C str.), 1642 (C=O amide), 1728 (C=O ester), 3010 (C-H Ar. str.), 3250 (NH).
1H-NMR (400 MHz, DMSO): 1.02-1.22 (4H, m, cyclohexyl-CH2), 1.11 (3H, t, J = 7.0 Hz, CH3-CH2),
1.25-1.43 (6H, br. s, cyclohexyl-CH2), 1.48-1.65 (4H, m, cyclohexyl-CH2), 1.65-1.90 (6H, m,
cyclohexyl-CH2), 3.11 (3H, s, N-CH3), 3.16 (2H, m, cyclohexyl-CH), 4.04 (2H, q, J = 7.0 Hz, CH2-
CH3), 4.61 (1H, s, CH2-S), 4.84 (1H, s, CH2-S), 5.14 (1H, d, J = 3.2 Hz, pyrimidine-CH), 7.25 (3H, dd,
13
J = 14.0, 7.0 Hz, Ar-H), 7.32 (2H, t, J = 7.0 Hz Ar-H), 8.10 (1H, d, J = 3.2 Hz, NH). C-NMR (100
MHz, DMSO): 13.9, 24.7, 25.7, 29.6, 30.6, 35.9, 52.3, 58.9, 60.2, 106.1, 126.2, 127.6, 128.6, 143.3,
146.8, 152.9, 164.8, 193.8. EI-MS: m/z (%): 529 (M+, 77). Anal. Calcd. for C28H39N3O3S2 (529.24): C,
63.48; H, 7.42; N, 7.93. Found: C, 63.50; H, 7.43; N, 7.94.
4.1.6.4. Ethyl 1-methyl-2-oxo-4-phenyl-6-((4-phenylpiperazine-1-carbonothioylthio)methyl)-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (20
)
Yield (0.34 g, 45%) as off-white solid with m.p. 104–105 °C. 1H-NMR (500 MHz, CDCl3): 1.18 (3H, t,
J = 7.1 Hz, CH3-CH2), 3.30 (3H, s, N-CH3), 3.32-3.47 (4H, m, piperazine-2CH2), 3.55-3.64 (4H, m,
piperazine-2CH2), 4.12 (2H, q, J = 7.1 Hz, CH2-CH3), 4.73 (1H, s, CH2-S), 4.88 (1H, s, CH2-S), 5.36
(1H, s, pyrimidine-CH), 5.41 (1H, s, NH), 7.22 (1H, m, Ar-H), 7.26-7.40 (4H, m, Ar-H), 7.46 (1H, m,
Ar-H), 7.49-7.53 (2H, m, Ar-H), 7.61-7.83 (2H, m, Ar-H). 13C-NMR (125 MHz, DMSO): 13.9, 29.6,
35.7, 49.6, 50.2, 52.5, 60.2, 106.1, 114.3, 121.8, 126.1, 127.6, 128.5, 129.0, 136.6, 143.3, 146.3, 152.9,
164.8, 193.4. EI-MS: m/z (%): 510 (M+, 91). Anal. Calcd. for C26H30N4O3S2 (510.18): C, 61.15; H,
5.92; N, 10.97. Found: C, 61.13; H, 5.95; N, 11.00.
4.1.6.5. Ethyl 6-((4-(2-ethoxyphenyl)piperazine-1-carbonothioylthio)methyl)-1-methyl-2-oxo-4-phenyl-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (21
Yield (0.39 g, 47%) as white solid with m.p. 110–111 °C. IR (KBr) (
)
υ
, cm-1): 857 (C-S str.), 1078 (C=S
1
str.), 1516 (C=C str.), 1635 (C=O amide), 1724 (C=O ester), 2983 (C-H Ar. str.), 3266 (NH). H-NMR
(500 MHz, CDCl3): 1.18 (3H, t, J = 7.1 Hz, CH3-CH2), 1.47 (3H, t, J = 6.9 Hz, CH3-CH2), 2.59-2.63
(4H, m, piperazine-CH2), 3.29 (3H, s, N-CH3), 3.31-3.47 (2H, m, piperazine-CH2), 3.82 (2H, m,
piperazine-CH2), 4.11 (2H, q, J = 7.1 Hz, CH2-CH3), 4.16 (2H, q, J = 6.9 Hz, CH2-CH3), 4.89 (1H, s,
CH2-S), 4.96 (1H, s, CH2-S), 5.37 (1H, d, J = 3.1 Hz, pyrimidine-CH), 5.42 (1H, d, J = 3.1 Hz, NH),
6.98-7.07 (3H, m, Ar-H), 7.23 (1H, d, J = 1.5 Hz, Ar-H), 7.26-7.34 (4H, m, Ar-H), 7.40 (1H, t, J = 7.7
Hz, Ar-H). 13C-NMR (125 MHz, DMSO): 13.9, 14.8, 29.6, 35.7, 49.5, 50.1, 52.5, 60.2, 63.4, 106.1,
113.0, 118.4, 120.9, 123.0, 126.2, 127.6, 128.7, 140.1, 143.3, 146.3, 151.2, 152.9, 164.7, 193.4. EI-MS:
m/z (%): 554 (M+, 62). Anal. Calcd. for C28H34N4O4S2 (554.20): C, 60.62; H, 6.18; N, 10.10. Found: C,
60.65; H, 6.21; N, 10.11.
4.1.6.6. Ethyl 6-((4-(4-methoxyphenyl)piperazine-1-carbonothioylthio)methyl)-1-methyl-2-oxo-4-phenyl-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (22
)
Yield (0.34 g, 42%) as white solid with m.p. 105–106 °C. 1H-NMR (400 MHz, DMSO): 1.12 (3H, t, J =
7.1 Hz, CH3-CH2), 3.14 (3H, s, N-CH3), 3.15 (4H, m, piperazine-CH2), 3.69 (3H, s, O-CH3), 4.05 (4H,
17

ACCEPTED MANUSCRIPT
m, piperazine-CH2), 5.18 (1H, s, pyrimidine-CH), 6.85 (2H, d, J = 8.1 Hz, Ar-H), 6.93 (2H, d, J = 8.1
13
Hz, Ar-H), 7.24-7.26 (3H, m, Ar-H), 7.29-7.34 (2H, m, Ar-H), 8.12 (1H, s, NH). C-NMR (100 MHz,
DMSO): 13.9, 29.6, 35.7, 49.6, 50.0, 52.5, 56.0, 60.2, 106.1, 115.1, 115.2, 126.2, 127.6, 128.6, 137.4,
143.3, 146.3, 152.8, 153.0, 164.7, 193.5. EI-MS: m/z (%): 540 (M+, 33). Anal. Calcd. for C27H32N4O4S2
(540.19): C, 59.98; H, 5.97; N, 10.36. Found: C, 59.99; H, 6.01; N, 10.37.
4.2. Biology
4.2.1. Preliminary in vitro anticancer screening
In vitro cytotoxicity of compounds 6, 7, 9-12, 15, 17–20 and 22 was evaluated according to the current
one-dose protocol of the National Cancer Institute (NCI, USA) assay against a panel of 60 tumor cell
lines at 10 µM concentration [37].
4.2.2. MTT assay for cytotoxicity
In vitro cytotoxicity (IC50) of compounds 13, 14, 16 and 21 was tested by using a standard colorimetric
method (MTT assay) against three human tumor cell lines including human prostate cancer (PC-3),
colorectal carcinoma (HCT-116) and breast cancer (MCF-7) obtained from ATCC via Holding company
for biological products and vaccines (VACSERA), Cairo, Egypt. Cell lines were cultured in RPMI-1640
medium with 10% fetal bovine serum. Penicillin and streptomycin were added at 37 °C. Cells were seed
in a 96-well plate at 37 °C for 48 h. After incubation the cells were treated with different concentration
of compounds and incubated for 24 h. After 24 h of drug treatment, 20 µL of MTT solution at 5 mg/mL
was added and incubated for 4 h. 100 µL of Dimethyl sulfoxide (DMSO) was added into each well to
dissolve the purple formazan formed. The colorimetric assay is measured and recorded at absorbance of
570 nm using a plate reader (EXL 800, USA). The relative cell viability in percentage was calculated as
(A570 of treated samples/A570 of untreated sample) x 100 [38].
4.2.3. Enzyme-Linked Immunosorbent Assay of caspase-3 and caspase-9
4.2.3.1. The Invitrogen Caspase-3 (active) Human ELISA assay
The number of 8-well strips needed for the assay was determined. The standard diluent buffer (100
was added to the zero standard wells, while chromogen blank was left empty. The standards, controls
and tested compound (100 L) were then added, and incubated for 2 hours at room temperature. Wells
were evacuated from liquids, and washed 4 times. Caspase-3 (active) detection antibody solution (100
L) was added to each well except the chromogen blank(s), and incubated for 1 hour at room
µL)
µ
µ
temperature. Again liquid was discarded from wells and washed 4 times. Anti-Rabbit IgG HRP working
solution was then added to each well except the chromogen blank and incubated for 30 minutes at room
temperature. Wells were washed 4 times. Stabilized Chromogen (100
µL) was added to each well, and
incubated for 30 minutes at room temperature. Finally, stop solution (100
µL) was added to each well
and the absorbance was read at 450 nm within 2 hours after addition. Curve fitting software was used to
generate the standard curve; from which the concentrations for tested compound and controls were read.
4.2.3.2. DRG® Caspase-9 (human) ELISA (EIA-4860) assay
First, cell lysates were prepared. The number of required microwell strips was determined and allowed
to be washed twice with wash buffer. The sample diluent was added in duplicate to the blank wells (100
µ
L) and to sample wells (50
µ
L). The sample was added in duplicate (50
µL) to designated sample
wells. Detection antibody (50
µ
L) was prepared and added to all wells, and incubated for 2 hours at
room temperature. Microwell strips were washed 3 times with wash buffer. Anti-rabbit-IgG-HRP was
18

ACCEPTED MANUSCRIPT
prepared and added (100
µL) to all wells, then incubated for 1 hour at room temperature. Microwell
strips were washed 3 times with wash buffer. TMB substrate solution (100
µ
L) was added, and
incubated for 10 minutes at room temperature. Finally, stop solution (100
intensity was measured at 450 nm.
µL) was added, and the color
4.2.4. Cell cycle analysis and induction of apoptosis
4.2.4.1. Flow cytometric analysis of cell-cycle distribution
For flow cytometric analysis of DNA content, A549 cells in exponential growth were treated with
compound 19 and incubated for 24 h. The cells were collected, centrifuged and fixed with ice cold
ethanol (70%). The cells were then treated with buffer containing RNAse A and 0.1% Triton X-100,
then stained with propidum iodide (PI) and compared with control which was treated with DMSO. DNA
contents were measured by flow cytometry.
4.2.4.2. Analysis of cellular apoptosis
A549 cells in exponential growth were treated with compounds 19 and incubated for 24 h. After an
incubation period, 1-5 x 105 cells were harvested and suspended in 500 µL of 1X Binding Buffer, 5 µL
of Annexin V-FITC and 5 µL of PI. They are incubated at room temperature for 5 min in the dark and
incubated at 37 °C. Annexin V-FITC conjugate is specially engineered to produce enhanced
fluorescence signal and photo stability. The Annexin V-FITC kit includes Annexin V-FITC for detecting
apoptosis and PI for detecting necrosis. Thus, Apoptosis and necrosis can be differentiated. Detection
and analyzing of Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) using FITC
signal detector (usually FL1) and PI staining by the phycoerythrin emission signal detector (usually
FL2). Visit http://www.biovision.com for details.
4.2.5. Enzyme activity inhibition assay
4.2.5.1. VEGFR-2 inhibition assay
The enzyme inhibition assay of compounds 14 and 19 against VEGFR-2 was evaluated using the
RayBio® Human VEGFR-2 ELISA (Enzyme-Linked Immunosorbent Assay) kit. The assay utilized a
specific antibody for human VEGFR-2 coated on a 96-well plate, where 100 µL of either standard
solution or tested compound was added to each well and incubated for 2.5 hours at room temperature.
Wells were washed; prepared biotin antibody (100 µL) was added and incubated for 1 hour at room
temperature. The unbound biotinylated antibody was washed away from each well; prepared
Streptavidin solution (100 µL) was added and incubated for 45 minutes at room temperature. Wells were
washed again followed by addition of TMB One-Step Substrate Reagent (100 µL) to each well and
incubated for 30 minutes at room temperature, where the color was produced proportionally to the
amount of bound VEGFR-2. Stop solution (50 µL) was added to each well and the color intensity was
read at 450 nm immediately. The mean absorbance for each set of duplicate standards controls and
tested compounds was calculated, and the average zero standard optical density was subtracted. The
standard curve was plotted on log-log graph paper with standard concentration on the X-axis and
absorbance on the Y-axis. % Inhibition was calculated via comparing results obtained from test
compounds with control, while IC50 was calculated from concentration/inhibition response curve
compared to sorafenib as a standard. Visit http://www.raybiotech.com for details.
4.2.5.2. mTor inhibition assay
19

ACCEPTED MANUSCRIPT
The enzyme inhibition assay of compounds 14 and 19 against mTOR was evaluated using Abcam’s
mTOR in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit. This assay employs
an affinity tag labeled capture antibody and a reporter conjugated detector antibody which
immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector
antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well.
To perform the assay, tested compounds or standards (50
included with the kit, followed by the antibody mix (50
compounds were assayed in duplicates. After incubation for 1 hour at room temperature, the wells were
washed with 3 x 350 L 1X Wash Buffer PT to remove unbound material. TMB substrate (100 L) was
added, incubated for 10 minutes in the dark, and then catalyzed by HRP, generating blue coloration.
This reaction was then stopped by addition of Stop Solution (100 L), shaken on a plate shaker for 1
µ
L) were added to the 96 well plate strips
µL). All standards, controls and tested
µ
µ
µ
minute to mix and complete any color change from blue to yellow. Signal was generated proportionally
to the amount of bound analyte and the intensity is measured at 450 nm. % Inhibition was calculated via
comparing results obtained from test compounds with control, while IC50 was calculated from
concentration/inhibition response curve compared to rapamycin as
a
standard. Visit
http://www.abcam.com for details.
5. Acknowledgment
We thank Dr. M. Youssif for his help during proofreading the article and Dr. George S. G. Shehatou for
his assistance in revision of biology section.
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Highlights
•
•
•
A new series of dihydropyrimidinones was designed and synthesized.
Cytotoxic activity was assessed against 60 cancer cell lines.
Compound 19 showed a significant activity against NCI-H460, SK-MEL-5, and HL-60(TB) cell
lines.
•
•
Compound 19 induced apoptosis and showed cell cycle arrest at G2/M phase.
Compounds 14 and 19 showed lower cytotoxic activity than doxorubicin against three normal
cells.
•
Compound 19 exhibited a 1.5-fold increase in mTOR enzyme activity over that of rapamycin.
22

Highlights
ACCEPTED MANUSCRIPT
• A new series of dihydropyrimidinones was designed and synthesized.
• Cytotoxic activity was assessed against 60 cancer cell lines.
• Compound 19 showed a significant activity against NCI-H460, SK-MEL-5, and HL-60(TB)
cell lines.
• Compound 19 induced apoptosis and showed cell cycle arrest at G2/M phase.
• Compounds 14 and 19 showed lower cytotoxic activity than doxorubicin against three normal
cells.
• Compound 19 exhibited a 1.5-fold increase in mTOR enzyme activity over that of rapamycin.